Sensitization Stokes shift

Fluorochromes have been introduced that offer excitation and emission spectra similar to those of fluorescein, but that overcome some of fluorescein s limitations. BODIPY FL has a short Stokes shift, but offers higher fluorescence intensity, and is claimed to be more photostabile and less pH-sensitive than fluorescein. Oregon Green 488 and the newly introduced Alexa 488 fluorochromes have spectra nearly identical to those of fluorescein, but are considerably more photostabile, and produce less quenching of fluorescence with higher numbers of fluorochromes per antibody than does fluorescein. 

HPTS is a highly water-soluble, pH-sensitive dye with a pK of 7.5 in aqueous solution [8], When in alkaline medium, pH > 7.5, acid-base equilibrium is totally displaced toward the anion form (3sPyO ) of the dye. The electronic character of 3sPyO remains unchanged after photo-excitation, and corresponds to a singlet-excited state [9], Fluorescence from this state undergoes a fast 0.4 ps Stokes shift and has a maximum at 515 nm and a lifetime of 5.3 0.1 ns [10],... 

The spectral characteristics of Lucifer Yellow iodoacetamide produce luminescence at somewhat higher wavelengths than the green luminescence of fluorescein, thus the yellow designation in its name. The excitation maximum for the probe occurs at 426 nm, and its emission at 530 nm. The rather large Stokes shift makes sensitive measurements of emission intensity possible without interference by scattered excitation-light. The 2-mercaptoethanol derivative of the fluorophore has an extinction coefficient at pH 7 of about 13,000 M-1cm-1 at 426 nm. 

Block the laser beam or spectrometer entrance slit and adjust the spectrometer to an anti-Stokes shift of 1000 cm Caution Exposure of the sensitive phototube to the intense Rayleigh scattering line can seriously damage the detector. Scan the anti-Stokes spectrum from 1000 to 150 cm in the parallel polarization configuration and, using appropriate sensitivity expansion, j measure the ratio of anti-Stokes to Stokes peak heights for each band. 

The choice of fluorophore has a considerable effect upon the sensitivity of the assay, and depends on the assay conditions. Ideally, the fluorophore should have a large Stokes shift (p. 233), it should be easily differentiated from endogenous fluorophores, and should be capable of being coupled simply to the drug. Fluorescein satisfies all these requirements and is the most popular fluorophore used. 

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